Muscarinic responses of gastric parietal cells

Summary Isolated rabbit gastric glands were used to study the nature of the muscarinic cholinergic responses of parietal cells. Carbachol (CCh, 100 m) stimulation of acid secretion, as measured by the accumulation of aminopyrine, was inhibited by the M1 antagonist, pirenzepine, with an IC₅₀ of 13 m; by the M2 antagonist, 11,2-(diethylamino)methyl-1 piperidinyl acetyl-5,11-dihydro-6H-pyrido 2,3-b 1,4 benzodiazepin-6-one (AF-DX 116), with an IC₅₀ of 110 m; and by the M1/M3 antagonist, diphenylacetoxy-4-methylpiperidinemethiodide (4-DAMP), with an IC₅₀ of 35nm. The three antagonists displayed equivalent IC₅₀ values for the inhibition of carbachol-stimulated production of¹⁴CO₂ from radiolabeled glucose, which is a measure of the turnover of the H,K-ATPase, the final step of acid secretion. Intracellular calcium levels were measured in gastric glands loaded with FURA 2. Carbachol was shown to both release calcium from an intracellular pool and to promote calcium entry across the plasma membrane. The calcium entry was inhibitable by 20 m La³⁺. The relative potency of the three muscarinic antagonists for inhibition of calcium entry was essentially the same as for inhibition of acid secretion or pump related glucose oxidation. Image analysis of the glands showed the effects of carbachol, and of the antagonists, on intracellular calcium were occurring largely in the parietal cell. The rise in cell calcium due to release of calcium from intracellular stores was inhibited by 4-DAMP with an IC₅₀ of 1,7nm, suggesting that the release pathway was regulated by a low affinity M3 muscarinic receptor or state; Ca entry and acid secretion are regulated by a high affinity M3 muscarinic receptor or state, inhibited by higher 4-DAMP concentrations (>30nm), suggesting that it is the steady-state elevation of Ca that is related to parietal cell function rather than the [Ca] _i transient. Displacement of³H N-methyl scopolamine (NMS) binding to purified parietal cells by CCh showed the presence of two affinities for CCh, but only a single affinity for 4-DAMP and lower affinity for pirenzepine and AFDX 116, providing further evidence for the parietal cell location of the [Ca] _i response. Elevation of steady-state [Ca] _i levels with either ionomycin or arachidonic acid did not replicate M3 stimulation of acid secretion or glucose oxidation, hence elevation of [Ca] _i is necessary but not sufficient for acid secretion.     

Cholinergic and adrenergic receptors on mouse cardiocytes in vitro

The effects of adrenergic and cholinergic receptor agonists and antagonists on single and clustered mouse cardiocytes in culture have been studied. Cardiocytes were obtained from mice, ranging in ages from 9 days in utero to 1 day postpartum, and were grown in culture for 2–14 days. Single isolated cells of every age tested possessed the ability to respond both via a muscarinic cholinergic receptor to the cholinergic agonist, carbamylcholine, and via α- and β-adrenergic receptors to norepinephrine and epinephrine. Thus, cholinergic and adrenergic receptors are simultaneously present on the same cell. Cardiocyte clusters had considerably higher sensitivity to both autonomic agents, but, because of the extensive functional specializations between cells, the localization of functional receptors to specific cells could not be made. [³H]Alprenolol, a potent β-adrenergic receptor antagonist, and [³H]quinuclidinyl benzilate ([³H]QNB), a potent muscarinic cholinergic receptor antagonist, were used to localize β-adrenergic and muscarinic cholinergic receptors by autoradiography. Quantitation of the muscarinic ACh receptor gave ～800 sites/μm², a value comparable to that for the nicotinic ACh receptor on primary skeletal muscle in culture. Electrophysiological and fine-structural studies confirmed the myocardial nature of these cells.     

Prostacyclin analogues inhibit canine parietal cell activity and cyclic AMP formation

To assess further the mechanism by which prostacyclin inhibits acid secretion, the actions of two stable prostacyclin analogues on parietal cell function and cyclic AMP formation were tested using enzymatically dispersed cells from canine fundic mucosa. Accumulation of ¹⁴C-aminopyrine (AP) was used as an index of parietal cell response to stimulation. The 16-phenoxy derivative of PGI₂ inhibited accumulation of AP stimulated by histamine (10 μM), with 50% inhibition (ID₅₀) at 10 nM. 6_β-PGI₁ also inhibited the action of histamine (ID₅₀ 0.5μM) but failed to block stimulation by carbachol or the dibutyryl derivative of cyclic AMP (dbcAMP). In similiar concentrations to those producing inhibition of histamine-stimulated AP accumulation, the 16-phenoxy analogue and 6_β-PGI₁ inhibited histamine-stimulated cyclic AMP generation by parietal cells. At 100 fold higher concentrations, 6_β-PGI₁ stimulated cyclic AMP formation, presumably in non-parietal cells. Even in high concentrations the 16-phenoxy analogue failed to increase cyclic AMP formation by mucosal cells. These data indicate that the stable prostacyclin analogues are potent, direct inhibitors of histamine-stimulated parietal cell function and that it is the inhibition, rather than the stimulation, of cyclic AMP formation that is linked to the antisecretory actions of these prostanoid compounds.     

The influence of magnesium on calcium-activated, vascular smooth muscle actomyosin ATPase activity

Histamine activation of adenylyl cyclase activity in sonicated enriched rat gastric parietal cells showed a time, temperature, and concentration dependence upon guanine diphosphoimide (Gpp(NH)p). Enzyme activation was first order with Gpp(NH)p alone or Gpp(NH)p plus histamine. The K_a for Gpp(NH)p was ~2 μm and was not influenced by histamine. GTP and GDP were inactive alone or with histamine and were competitive with Gpp(NH)p, showing apparent K_i's of near 0.4 and 0.3 μm, respectively. In the presence of Gpp(NH)p, parietal cell adenylyl cyclase was activated by histamine with an EC₅₀ of 24 μm, the most potent in a series of histamine analogs, further substantiating an H₂-receptor classification for this response. H₂-Receptor antagonists were competitive inhibitors with submicromolar K_i's. Preincubation of parietal cells with histamine and Gpp(NH)p resulted in adenylyl cyclase activity up to 15 times the basal level. The activated state was retained after washing the cells free of histamine and Gpp(NH)p and was not reversed by the subsequent addition of either histamine, cimetidine, or GTP. The other gastric acid secretagogues, pentagastrin and carbamylcholine, were without effect upon histamine activation or the activated state of adenylyl cyclase. These results describe a level of control of histamine-sensitive adenylyl cyclase that requires consideration in the activation of the parietal cell H₂-receptor system by histamine to modulate acid secretion.     

Genetic Ablation of the ClC-2 Cl- Channel Disrupts Mouse Gastric Parietal Cell Acid Secretion

The present studies were designed to examine the effects of ClC-2 ablation on cellular morphology, parietal cell abundance, H/K ATPase expression, parietal cell ultrastructure and acid secretion using WT and ClC-2^-/- mouse stomachs. Cellular histology, morphology and proteins were examined using imaging techniques, electron microscopy and western blot. The effect of histamine on the pH of gastric contents was measured. Acid secretion was also measured using methods and secretagogues previously established to give maximal acid secretion and morphological change. Compared to WT, ClC-2^-/- gastric mucosal histological organization appeared disrupted, including dilation of gastric glands, shortening of the gastric gland region and disorganization of all cell layers. Parietal cell numbers and H/K ATPase expression were significantly reduced by 34% (P<0.05) and 53% (P<0.001) respectively and cytoplasmic tubulovesicles appeared markedly reduced on electron microscopic evaluation without evidence of canalicular expansion. In WT parietal cells, ClC-2 was apparent in a similar cellular location as the H/K ATPase by immunofluorescence and appeared associated with tubulovesicles by immunogold electron microscopy. Histamine-stimulated [H⁺] of the gastric contents was significantly (P<0.025) lower by 9.4 fold (89%) in the ClC-2^-/- mouse compared to WT. Histamine/carbachol stimulated gastric acid secretion was significantly reduced (range 84–95%, P<0.005) in ClC-2^-/- compared to WT, while pepsinogen secretion was unaffected. Genetic ablation of ClC-2 resulted in reduced gastric gland region, reduced parietal cell number, reduced H/K ATPase, reduced tubulovesicles and reduced stimulated acid secretion.     

Localization of histamine and histamine H2-receptor antagonists in the gastric mucosa.

Synopsis Histamine stimulates acid secretion by the parietal cell and this secretion is inhibited by the histamine H₂-receptor antagonists. Whole body autoradiography showed that radioactivity from¹⁴C-histamine was localized in the artery walls of the stomach and in the muscularis mucosae, but that the level in the fundic mucosa was the same as the blood.When the H₂-receptor antagonists burimamide, metiamide and cimetidine were labelled with³⁵S,¹⁴C or³H and dosed to rats, whole body autoradiography showed that the stomach was predominantly labelled in the glandular mucosa from 5 to 120 min after administration. Microautoradiography in the rat and dog after intravenous injection of [³H] metiamide or [³H] cimetidine demonstrated an uptake of tritium in the parietal cell cytoplasm that was 3- to 4-times greater than that found in adjacent peptic cells or areas of muscularis mucosa. The preferential labelling persisted at a low level up to 6 h after injection in the rat. The localization of radioactivity from the H₂-antagonists in the parietal cell cytoplasm correlates well with their pharmacological activity in preventing acid secretion from this cell.     

Role of Rho in rabbit parietal cell

Rho is known as an important regulator of actin microfilament formation. We were led to study it because a dynamic rearrangement of actin filaments occurs during activation of gastric acid secretion. In order to use specific probes, the rabbit gastric gland culture system was employed and the various genes were expressed using adenovirus vector. When the constitutive active mutant of Rho (RhoAV14) was expressed, histamine- or carbachol-stimulated acid secretion monitored by (14)C-aminopyrine accumulation was inhibited. Conversely, expression of C3 toxin, the specific inhibitor of Rho, and expression of G(12/13)-specific regulator of G-protein signaling domain, the specific inhibitor of G(12/13) which is considered to be an upstream mediator of Rho, both potentiated acid secretion stimulated by the agonists. F-actin staining of parietal cell expressing RhoAV14 revealed that the microfilament supporting the intracellular canaliculi (not on the basolateral membrane) almost disappeared. No clear changes in the intracellular localization of Rho were observed during stimulation of parietal cell. In resting glands, the endogenous active form of Rho was relatively high, and it decreased during histamine stimulation. The finding that any treatment which inhibit Rho augment acid secretion whereas those that activate Rho inhibit secretion strongly suggests that the Rho-pathway conducts a negatively regulating signal in parietal cell activation, possibly via site-specific regulation of actin microfilaments.     

Maturation of muscarinic agonist receptors in rat developing pancreas and its relation to maximal enzyme secretion

The true K_Ds of [³H] (?) QNB binding to muscarinic receptors were found to be 4.13 and 6.43 × 10^?11 M in pancreas of 21 day fetal and adult rats. The competition curves of specific [³H] (?) QNB bound by two antagonists have shown that the affinity of these drugs did not change with age with Hill coefficients near unity. However, with the agonist carbamylcholine as the competitive drug, a more flat curve was obtained with a Hill coefficient below unity. At least two populations of carbamylcholine binding sites were found with different K_Ds: a K_H around 7 × 10^?7 M and a K_L around 3 × 10^?5 M. These two populations were present during all the developmental periods studied. The ED₅₀ of bethanechol stimulated amylase secretion did not change within the age limit studied (from 11 to 365 days). The high affinity sites for carbamylcholine would seem to be the receptor implicated in the physiological response of the pancreas.     

Nicotinic acetylcholine receptors on a cholinergic nerve terminal in the cockroach,Periplaneta americana

Summary Intracellular microelectrode recording and ionophoretic application of carbamylcholine (CCh) were used to compare the cholinergic sensitivity of postsynaptic dendrites of an identified neurone with that of an identified presynaptic cholinergic axon.The axon of the lateral filiform hair sensory neurone (LFHSN) in the first-instar cockroachPeriplaneta americana was found to be as sensitive to CCh as the dendritic regions of giant interneurone 3 (GI 3). The CCh response of both neurones was unaffected by replacing Ca²⁺ with Mg²⁺, confirming that the ACh receptors are present on the neurones under test. The CCh response of both neurones was mimicked by ionophoretic application of nicotine. The responses were blocked by 10^–5 M mecamylamine and 10^–6 M d-tubocurarine and were not affected by muscarinic antagonists, suggesting that the ACh receptors present on GI 3 and LFHSN are predominantly nicotinic.The muscarinic agonist oxotremorine and the antagonists atropine and quinuclidinyl benzilate had no modulatory effect on LFHSN-GI 3 synaptic transmission.The latency of the LFHSN response to CCh was consistent with the hypothesis that ACh receptors are situated on the main axon/terminal within the neuropil of the ganglion. It has previously been shown that this region of the axon does not form output synapses (Blagburn et al. 1985a). This indirect evidence indicates that presynaptic or extrasynaptic ACh receptors are present in the membrane of a cholinergic axon.LFHSN was depolarized by synaptically-released ACh after normal or evoked spike bursts, suggesting that the nicotinic ACh receptors act as autoreceptors. However, it was not possible to obtain direct evidence to support the hypothesis that these receptors modulate ACh release.Abbreviations CCh carbamylcholine - GI giant interneurone - FHSN filiform hair sensory neurone - LFHSN lateral filiform hair sensory neurone - R _in input resistance - V depolarization - V _m resting potential     

Potassium release from submandibular salivary gland in vitro

Rat submandibular gland slices, incubated in continuously-gassed Krebs-Ringer bicarbonate buffer, were shown to release K⁺ in response to α-adrenergic and muscarinic cholinergic stimulation. The system employed the specific α-, β-adrenergic and cholinergic receptor-blocking agents phentolamine, propranolol and atropine, respectively, in combination with the agonists l-epinephrine and carbamylcholine both of which required the presence of Ca²⁺ for their effect. The introduction of Ca²⁺ into the cell via the ionophore A23187, with all neurotransmitter receptors blocked, resulted in K⁺ release. Ouabain also allowed extensive K⁺ release which was in addition to, and hence independent of, that elicited by epinephrine and carbamylcholine. Ethacrynic acid, a potent inhibitor of salivary secretion in vivo, had no influence on K⁺ movement. K⁺ was released by both physalaemin and an eledoisin-related peptide independently of normal neurotransmitter receptors. The activity of the eledoisin-related peptide did not require the presence of extracellular Ca²⁺. The implication of cyclic GMP at some stage of K⁺ release was suggested by experiments with a phosphodiesterase inhibitor.The results support an hypothesis where the initial stimulus at either α-adrenergic or muscarinic cholinergic receptors causes an immediate permeability change such that Ca²⁺ enters the cells resulting in K⁺ release. The loss of K⁺ is quickly countered by the ouabain-sensitive ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$) ATPase which would be activated by the lowered intracellular K⁺ levels.     

Studies on adenyl cyclase in Necturus gastric mucosa

Adenyl cyclase activity of Necturus gastric mucosa was determined by measuring the amount of radioactive adenosine 3′,5′-cyclic monophosphate formed from ³H-labeled adenosine triphosphate. Histamine, pentagastrin, and fluoride added in vitro significantly increased fundic adenyl cyclase activity. The dose response curves show that the affinity of pentagastrin for adenyl cyclase is greater than that of histamine, whereas the peak response to pentagastrin is less than that to histamine. Additive stimulation was not obtained when maximal doses of pentagastrin and histamine were combined. These findings suggest that gastric mucosa contains a single adenyl cyclase unit which is coupled to distinctive selectivity sites for gastrin and histamine. N-Benzyl-3-pyrrolidyl acetate methobromide (AHR-602), the muscarinic compound, caused a significant reduction in adenyl cyclase activity, indicating a different mechanism of stimulation of gastric acid secretion for cholinergic muscarinic compounds.     

Reconstitution of solubilized atrial cholinergic muscarinic receptors in liposomes

The reconstitution of solubilized bovine atrial cholinergic muscarinic receptor into liposomes made of exogenous lipids has been achieved by polyethyleneglycol precipitation. Of the different lipid mixtures used, soybean lecithins were shown to be the best on the basis of receptor recovery. The receptor reconsituted into soybean lecithins liposomes exhibited ligand binding properties very similar to those of the native receptor. The dissociation constant of [³H]-N-methyl-scopolamine ([³H]NMS) was 0.46 and 0.30 nM as determined by equilibrium and kinetics experiments respectively. The potency of a range of muscarinic ligands in displacing [³H]NMS binding was atropine > methyl-atropine > scopolamine > pirenzepine oxotremorine > gallamine > carbamylcholine > pilocarpine bethanechol. The Hill slopes of the displacement curves were near 1 for the antagonists and smaller than 1 for the agonists and for gallamine. The agonist binding may be modulated by guanine nucleotides. These results indicate that soybean lecithins fulfill the lipid requirements for the reconstitution of the atrial muscarinic receptor.     

Increased Insulin Secretion by Muscarinic M1 and M3 Receptor Function from Rat Pancreatic Islets in vitro

Parasympathetic system plays an important role in insulin secretion from the pancreas. Cholinergic effect on pancreatic beta cells exerts primarily through muscarinic receptors. In the present study we investigated the specific role of muscarinic M1 and M3 receptors in glucose induced insulin secretion from rat pancreatic islets in vitro. The involvement of muscarinic receptors was studied using the antagonist atropine. The role of muscarinic M1 and M3 receptor subtypes was studied using subtype specific antagonists. Acetylcholine agonist, carbachol, stimulated glucose induced insulin secretion at low concentrations (10⁻⁸–10⁻⁵ M) with a maximum stimulation at 10⁻⁷ M concentration. Carbachol-stimulated insulin secretion was inhibited by atropine confirming the role of muscarinic receptors in cholinergic induced insulin secretion. Both M1 and M3 receptor antagonists blocked insulin secretion induced by carbachol. The results show that M3 receptors are functionally more prominent at 20 mM glucose concentration when compared to M1 receptors. Our studies suggest that muscarinic M1 and M3 receptors function differentially regulate glucose induced insulin secretion, which has clinical significance in glucose homeostasis.     

Histamine H2-receptors in the gastric mucosa: role in acid secretion.

Currently, two major hypotheses dominate thinking about the role of histamine in the regulation of gastric acid secretion. Code has proposed that histamine is the final common mediator of secretagogue action on the parietal cell while Konturek and Grossman have suggested a multi-receptor control of the secretory process. Experimental results derived from the use of recently synthesized histamine H₂-receptor antagonists have been used by both groups to support their hypotheses. Paradoxically, these hypotheses depend on the presumed specificity of the H₂-antagonists in blocking histamine mediated acid secretion while the apparent lack of such secretagogue specificity of the H₂-antagonists is an important basis for the development of the hypotheses. Our review will analyze the experimental evidence which implicates the histamine H₂-receptor in the control of hydrogen ion secretion as well as evidence for and against receptor specificity in the gastric mucosa of histamine H₂-receptor antagonists.     

Cholinergic-stimulated alkaline phosphatase secretion and phospholipid synthesis in guinea pig seminal vesicles

Incubation of slices of seminal vesicles of the guinea pig with cholinergic drugs led to an enhanced secretion of alkaline phosphatase. Incubation with carbamylcholine also stimulated the incorporation of P³² into the phospholipid fraction. Both cholinergic effects required a supply of energy since dinitrophenol was inhibitory. The stimulation of enzyme secretion and phospholipid synthesis by carbamylcholine was completely abolished by atropine. Omission of calcium ions from the incubation medium and pre-treatment of the slices with ethylenediaminetetraacetic acid (EDTA) caused a marked reduction in alkaline phosphatase secretion induced by carbamylcholine but had no effect on incorporation of P³² into phospholipids. Adenergic agents such as epinephrine, norepinephrine and isoproterenol did not influence these two processes. Addition of cyclic AMP, dibutyryl cyclic AMP and a phosphodiesterase inhibitor was also ineffective. The incorporation of P³² into the various phospholipids of the seminal vesicle was examined. In the presence of carbamylcholine, there was a marked increase in the P³²-specific activities of phosphatidylinositol (nearly 6-fold) and of phosphatidylserine (about 1.5-fold). These observations indicate that the guinea pig seminal vesicle, a large hollow organ composed of a single layer of epithelium, is ideally suited for studies concerning the biochemistry of macromolecular secretion.     

Purification and molecular properties of the acetylcholine receptor from Torpedo electroplax

The acetylcholine receptor of Torpedo electroplax is purified by affinity adsorption using cobra toxin (Naja naja siamensis) covalently attached to Sepharose 4B. Desorption by 10 mm benzoquinonium produces a protein that binds α-[¹²⁵I]bungarotoxin but not [³H]acetylcholine or other reversible cholinergic ligands. On the other hand, desorption by 1 m carbamylcholine produces an acetylcholine receptor protein that binds [³H]acetylcholine, [³H]decamethonium, [³H]nicotine, [¹⁴C]dimethyl-d-tubocurarine, and α-[¹²⁵I]bungarotoxin. The batch method of affinity adsorption employed gives recoveries of acetylcholine receptor (as measured by acetylcholine binding) averaging 69.2 ± 14.6%. The purity of the isolated acetylcholine receptor protein is estimated to be at best 87% as judged by disc gel electrophoresis and electrofocusing.The purified acetylcholine receptor binds 7.8 nmoles acetylcholine/mg protein based on estimation of protein concentration by a spectrophotometric method. Of these, 2.7 nmoles exhibit high affinity (K_D = 0.02 μM) and 5.1 nmoles a lower affinity (K_D = 1.97 μM. If the protein concentration used is that obtained by amino acid analysis, the total specific activity would be 10.4 nmoles acetylcholine bound per milligram protein. The subunit carrying one acetylcholine binding site is estimated to range between 83,000 and 112,000 daltons. In contrast to the membrane-bound or Lubrol-solubilized acetylcholine receptor, the purified acetylcholine receptor shows no autoinhibition with acetylcholine concentrations up to 10 μm. Binding of acetylcholine was totally inhibited by α-bungarotoxin or cobra toxin and was partially blocked by four nicotinic drugs, but not by two muscarinic ones. The amino acids of the acetylcholine receptor are analyzed and compared to those of acetylcholinesterase.     

The adenylate cyclase-cyclic AMP-protein kinase system in different cell populations of the guinea pig gastric mucosa

In isolated guinea pig gastric mucous and enriched parietal cells it was tested whether or not cyclic AMP in response to histamine stimulation might reach concentrations sufficiently high to activate an intracellular cyclic AMP-dependent protein kinase and thereby mediate the acid response. Although histamine stimulated parietal cell adenylate cyclase to a greater extent than mucous cell adenylate cyclase, cyclic AMP levels in response to maximal histamine stimulation reached higher levels in mucous than in parietal cells. This had to be attributed to a five times higher phosphodiesterase activity in parietal cell than in mucous cell populations. In the absence of the phosphodiesterase inhibitor isobutylmethylxanthine exposure of the cells to histamine only in mucous cells produced an increase in cyclic AMP-dependent protein kinase activity ratio, but not in parietal cells. Dibutyryl-cyclic AMP induced cyclic AMP accumulation in parietal cell populations was compared to dibutyryl-cyclic AMP induced H+ secretion, as measured by 14C-aminopyrine uptake. A maximal acid response was associated with an intracellular cyclic AMP level of approximately 300 pmol/10(6) cells, which was never reached by maximal histamine stimulation even not in the presence of the phosphodiesterase inhibitor. It is concluded that activation of the parietal cell cyclic AMP-dependent protein kinase is one way for stimulating H+ secretion, but that the acid response elicited by histamine requires another intracellular pathway.     

Muscarinic and nicotinic receptor-mediated Ca2+ dynamics in rat adrenal chromaffin cells during development

To clarify when the cholinergic receptor-mediated secretion mechanism of developing adrenal chromaffin cells is expressed and becomes functional, morphological changes and intracellular calcium dynamics were studied by immunohistochemistry, electron microscopy, and Fura-2 digital image analysis. From embryonic day 14 to 16, adrenal medullary cells were immunoreactive to noradrenaline-synthesizing enzyme (dopamine β-hydroxylase) but not to adrenaline-synthesizing enzyme (phenylethanolamine N-methyltransferase). These cells contained either no granules or just a few granules of high electron density. Exocytotic figures were rarely observed in cells of the control or in cells after carbamylcholine stimulation. Nerve fibers in the adrenal medulla contained either no clear vesicles or very few. Neither methacholine nor nicotine caused a change of intracellular Ca²⁺ in most chromaffin cells. From embryonic day 18 to 20, chromaffin cells were immunoreactive to both dopamine β-hydroxylase and phenylethanolamine N-methyltransferase and they contained relatively numerous secretory granules. Exocytotic figures were often seen in cells after carbamylcholine stimulation. The intra-adrenal nerve fibers contained numerous clear vesicles and a few dense-cored vesicles. Methacholine caused no rise of intracellular Ca²⁺, but nicotine induced a low to relatively high rise in many cells. From postnatal day 2 or 3 to postnatal week 1, numerous cells were immunoreactive to both dopamine β-hydroxylase and phenylethanolamine N-methyltransferase, whereas some cells were reactive to dopamine β-hydroxylase alone. Chromaffin cells were divisible into noradrenaline cells and adrenaline cells based on the ultrastructural features of their granules. Methacholine induced a moderate rise of intracellular Ca²⁺ and nicotine caused a high rise in many chromaffin cells, whereas, in some chromaffin cells, methacholine induced no rise of intracellular Ca²⁺ and nicotine induced a high rise. These results suggest that morphological changes of the developing cells and the intra-adrenal nerve fibers are related to the expression of a cholinergic receptor-mediated secretion mechanism and that this mechanism via a nicotinic receptor-mediated Ca²⁺ signaling pathway precedes the muscarinic receptor-mediated one during development.     

Muscarinic receptor sites in human foetal brain

The specific binding of the muscarinic cholinergic ligand N-methylscopolamine to human foetal brain has been measured. A level of binding of 64 pmol/g protein was found with a dissociation constant, K_d of 0.27 nM. Values of 0.17 nM min^?1 and 0.048 min^?1 for the association rate constant, K_on, and the dissociation rate constant K_off respectively, were obtained. The pharmacological properties of the binding site were found to be very similar to those reported for muscarinic receptors from adult mammalian brain except that the binding of pirenzepine and the nicotinic antagonists d-tubocurarine and decamethonium shows differences from that seen in adult brain.     

Phosphorylation of the α-subunit of (Na+ + K+)-ATPase by carbachol in tissue slices and the role of phosphoproteins in stimulus-secretion coupling

This study examined the changes in protein phosphorylation in response to cholinergic (muscarinic) stimulation of salivary secretion in the rat submandibular gland. Carbachol stimulation was associated with phosphorylation in a number of protein bands as detected by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and autoradiography. The molecular masses (M_r) of two proteins, in which the amount of phosphorylation more than doubled in response to carbachol, were 22 000 and 96 000. The M_r 96 000 protein precipitated at 120 000 × g while most of the M_r 22 000 protein remained in the supernatant at this speed. The effect of carbachol on the phosphorylation of the M_r 22 000 and 96 000 proteins was blocked by atropine, indicating that the cholinergic receptor involved is muscarinic. The time course of phosphorylation of the M_r 22 000 protein consisted of a rapid incrase in phosphorylation within the first min of carbachol stimulation. This increased phosphorylation persisted for less than 1 min. The increased phosphoryaltion of the M_r 96 000 protein also occurred within the first min but it persisted for at least 10 min. However, removal of the muscarinic agonist, carbachol, resulted in the rapid dephosphorylation of this protein. When the plasma membranes were purified, the M_r 96 000 protein was phosphorylated by ATP in the presence of Na⁺ and Mg²⁺. It was dephosphorylated by K⁺. This proves that the M_r 96 000 dalton protein is the α-subunit of the (Na⁺ + K⁺)-ATPase.